Ovarian estrogens exert critically important homeostatic feedback actions in the brain, pituitary, and ovary to restrain androgen biosynthesis. Disruption of these feedback controls invariably leads to dysregulation of gonadotropin secretions, androgen excess, and impaired fertility. Estrogen receptor alpha (ERa) appears to mediate most of these major negative feedback actions. ERa signaling mechanisms include classical genotropic effects mediated by direct binding of receptors to DNA, non-classical genotropic effects involving tethering of ERs to other transcription factors, and non-classical non-genotropic actions mediated by ERs coupled to membrane-initiated signaling pathways. The proposed studies use novel ERa mutant mice to further determine cellular mechanisms by which ERa mediates E2 feedback effects. We have utilized mutant ERa knock-in mice, which confer non-classical genotropic and non-genotropic signaling in the absence of classical signaling, to determine that non-classical ERa signaling conveys E2 negative feedback actions on LH secretion. Aim 1 determines if these feedback actions are manifest in brain by directly monitoring GnRH secretory responses to E2 treatments in WT (ERa+/+), ERa gene knockout (ER-/-), and non-classical ERa gene knock-in (ER-/AA) mice, as well as in neuron-specific ERa-/- mice. Aim 2 examines the involvement of non-classical genotropic vs. non-genotropic ERa signaling in negative feedback; the ability of E2 to exert actions in afferent circuitries controlling GnRH release will be compared among the three genotypes. Aim 3 analyzes the locus of intraovarian feed-back in theca cell-specific ERa null mutant mice. We then assess involvement of classical vs. non-classical ERa signaling in regulating androgen synthesis by analyzing E2 effects in cultured ovarian follicles of ERa+/+, ERa/-., ERa-/AA mice. In Aim 4, we determine the extent to which E2 actions are mediated by non-classical genotropic vs. membrane-initiated ERa signaling. New animals will be generated that express ERs conferring non-classical membrane-initiated signaling only, or non-classical genotropic signaling only. The extent to which E2 actions in the reproductive axis are rescued in these mice will be determined. These experiments will clarify the basic mechanisms by which ERa signaling regulates cellular and physiological function, and may provide important insights into the pathogenesis of hyperandrogenism in endocrine disorders such as polycystic ovary syndrome (PCOS).